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. 2012 Mar;86(5):2769-79.
doi: 10.1128/JVI.05657-11. Epub 2011 Dec 14.

Viral interferon regulatory factors are critical for delay of the host immune response against rhesus macaque rhadinovirus infection

Bridget A Robinson  1 , Megan A O'Connor, He Li, Flora Engelmann, Britt Poland, Richard Grant, Victor DeFilippis, Ryan D Estep, Michael K Axthelm, Ilhem Messaoudi, Scott W Wong
Affiliations

Viral interferon regulatory factors are critical for delay of the host immune response against rhesus macaque rhadinovirus infection

Bridget A Robinson et al. J Virol. 2012 Mar.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host.

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Figures

Fig 1
Fig 1
Detection of RRV DNA and lytic replication measured in RRV-infected RMs. Expanded-specific-pathogen-free RMs were infected intravenously with 5 × 106 PFU of WTBAC RRV (n = 6) or vIRF-ko RRV (n = 8). Each RM is labeled with an identifying number and an individual symbol. (A and B) Whole blood was analyzed weekly via qPCR to determine RRV loads. Data are represented as number of RRV genome copies/100 ng DNA in each of the WTBAC RRV-infected RMs (A) and the vIRF-ko RRV-infected RMs (B). (C and D) PBMCs (2 × 105) were cocultured with confluent RFs and serially diluted (1:3) across a 24-well plate. Viremic score correlates with the highest dilution of PBMCs that resulted in CPE when cocultured with RFs. A viremic score of 5 indicates the presence of CPE in wells with the highest dilution of PBMCs (2.5 × 103 PBMCs), and the presence of CPE in duplicate wells at each dilution has a viremic score of 0.5. (E) The presence of RRV DNA in cocultures was verified via nested PCR using primers for RRV ORF R3 (vMIP), as well as RRV ORF R10 (vIRF), to differentiate WT and vIRF-ko RRV.
Fig 2
Fig 2
Decreased B cell hyperplasia in vIRF-ko RRV-infected RMs. (A) Absolute numbers of CD20+ cells were calculated by converting the percentage of CD20+ lymphocytes using the total number of lymphocytes/ml blood obtained via a complete blood count machine (Hemavet). Data are represented as absolute number (106) of CD20+ cells/ml peripheral blood and are averaged (± SEM) among the WT RRV-infected cohort (n = 6) and the vIRF-ko RRV-infected cohort (n = 8). (B) The average (± SEM) frequency of Ki67+ memory B cells was calculated for WTBAC and vIRF-ko RRV-infected animals as described for panel A, and the baseline (preinfection) Ki67+ population was set equal to 1. Data were analyzed using an unpaired Student's t test. **, P ≤ 0.01; *, P ≤ 0.05.
Fig 3
Fig 3
Lower levels of viral persistence in vIRF-ko RRV-infected RMs. The presence of RRV genomic DNA within CD20+ cells was analyzed between 3 and 24 months postinfection. CD20+ B cells were sorted from PBMCs, DNA was extracted, and real-time qPCR (A) and nested PCR (B) analyses were performed to detect RRV ORF R3 (vMIP) and ORF R10 (vIRF).
Fig 4
Fig 4
CD4 and CD8 T cell proliferation in peripheral blood and bronchoalveolar lavage fluid. The frequency of proliferating (Ki67+) CD4 and CD8 T cells within PBMCs (A and B) and BAL fluid cells (C and D) was determined by flow cytometry. Preinfection samples were used to establish a baseline, and the fold change in Ki67+ populations in each animal was calculated for each time point. Data are presented as an average (± SEM) of the WTBAC RRV-infected RMs and the vIRF-ko RRV-infected RMs.
Fig 5
Fig 5
Infection of RMs with vIRF-ko RRV initiates an earlier RRV-specific T cell response. The frequency of RRV-specific T cells within PBMCs was determined after ex vivo stimulation with RRV overnight, followed by surface staining for CD4 and CD8 and intracellular cytokine staining for IFN-γ and TNF-α. IFN-γ single-positive (IFN-γ+) and double-positive (IFN-γ+/TNF-α+) cells are added into the final, calculated response. (A) Responses measured at 7 dpi in CD4 and CD8 T cells were compared via unpaired t test. (B to E) T cell responses measured during the first 63 dpi are represented in graphs containing data from WTBAC RRV-infected RMs on the left (B and D) and graphs containing data from vIRF-ko RRV-infected RMs on the right (C and E). Data are presented as percentage of RRV-responsive T cells, with baseline and nonspecific (VV) responses subtracted. Median responses are displayed as horizontal lines at each time point. Early time points are boxed to indicate statistical trends and significance and are identical to the data shown in panel A.
Fig 6
Fig 6
Anti-RRV IgG responses in RMs infected with WTBAC RRV or vIRF-ko RRV. IgG endpoint titers in the plasma were determined using an RRV-specific ELISA. Data are represented as an average (± SEM) of WTBAC RRV-infected RMs (n = 6) and vIRF-ko RRV-infected RMs (n = 8).
Fig 7
Fig 7
Cytokine levels in the plasma of RRV-infected RMs. Plasma cytokine levels were measured using a rhesus macaque-specific Milliplex kit: IFN-γ (A and B), IL-12p40 (C and D), and IL-18 (E and F). Cytokine responses measured prior to infection were subtracted from the responses at subsequent time points, and data are graphed as pg/ml, with median responses represented as horizontal bars at each time point. WTBAC RRV-infected RMs are graphed on the left, and vIRF-ko RRV-infected RMs are graphed on the right.
Fig 8
Fig 8
Measuring biologically active type I IFN in the plasma of RRV-infected RMs. Biologically active IFN-α was measured in plasma from WTBAC RRV-infected RMs (A) or vIRF-ko RRV-infected RMs (B) using a luciferase reporter assay. At the time points indicated, plasma was added to cells constitutively expressing firefly luciferase under an ISRE promoter, tRF-ISRE. Baseline readings (preinfection) were subtracted from readings at all subsequent time points. Data were normalized to constitutively expressed Renilla luciferase. The number of animals with measured IFN responses was calculated at each time point, and the dichotomous data set (no response = 0, response = 1) was compared using logistical regression. Using responses from the WTBAC RRV-infected animals as the reference (WT = 0, vIRF-ko = 1), virus (P = 0.0014; OR, 5.07; 95% CI, 1.87 to 13.72) and dpi (P = 0.0085; OR, 1.17; 95% CI, 1.04 to 1.32) were set as independent variables. ISRE, interferon-stimulated response element; RLU, relative luciferase units.
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